1a.Objectives (from AD-416)
Evaluate utility of small quantity malting and malt analysis methodology. Tocochromanols (vitamin E) are a group of amphiphilic lipid compounds produced exclusively in photosynthetic organisms. Although largely assumed to function in ameliorating the damaging effect of reactive oxygen species principally in green tissue, there is evidence that tocols perform other physiological roles. A recent report showed Arabidopsis mutants, deficient in tocopherol biosynthesis, were retarded in growth compared to wild type when subjected to low temperature treatment. They also did not export photoassimilate to sink tissues under low temperature conditions as well as wild types.
It might, therefore, be of interest to evaluate several lines of winter barley vs spring barley for their leaf tissue tocol composition to determine if varieties adapted to these growth habits present demonstrable differences.

1b.Approach (from AD-416)
Current methodology for evaluating the malting quality of a barley selection or line generally requires 150 – 200 g of barley of that line in order to obtain a representative malting and to provide sufficient material for subsequent analysis. The requirement for these quantities of grain for an initial malt analysis forces breeding programs to analyze only relatively advanced or to limit evaluations to a limited set of parameters on unmalted barley. We have modified the primary QA analyses to require significantly less material, while retaining analytical sensitivity and discrimination. A preliminary report suggested that representative maltings could be achieved with < 2 g of barley. Combining the modified malting and malt analysis methodologies could enable earlier-generation malt quality analysis, potentially shortening the breeding cycle by one or more years. The objective of this project is to conduct a significant test of the new methodology by malting and analyzing 1000 – 1500 barley lines using the newly-developed procedures and comparing the QA results with those from our standard malting and QA protocols on the same submissions.
We will select approximately 1000 from breeders submissions and variety checks that have been submitted to CCRU for malt quality evaluation. Our intent is to sample widely, from both Midwest and Western programs, 6-row and 2-row, early-stage selections and advanced lines, as well as accepted malting varieties. We will place 1 – 2 g of each barley line in a 1.75” diameter wire mesh “tea ball”, and bury the tea balls inside additional carrier check malt (6-row or 2-row as appropriate) in 1 kg containers in one of two Joe White micromalters available in our lab. After steeping to 46% moisture, germinating and kilning following standard laboratory protocols, and cleaning rootlets and acrospires from the kilned malt, we will grind the malt using a MiniBeadBeater-96 ball mill, sample and mash triplicate 187.5 mg aliquots, and analyze the resulting worts for extract (Anton-Paar DMA 4500M) or extract calculated from refractive index (Mettler RE-50), soluble protein (Nanodrop 2000-C or equivalent), wort ß-glucan and FAN (unpublished thermocycler method developed in our lab), and compare the mean results and sample variability with results from the traditional system both for accuracy of the mean result as well as sample variability.
We expect that this will be a rigorous, informative evaluation, showing both strengths and weaknesses of the reduced-quantity methodology and approach. If the results from the comparison are acceptable, this will add an attractive alternative to our standard QA analysis service for limited-quantity samples. Ultimately, this may speed up breeding line selections as well as simplifying analysis of specialized genetic populations.
Several winter and spring cultivars, grown in growth chambers under a range of temperature conditions, will be analyzed for tocols. Tocols will be extracted by published methods and analyzed by normal phase HPLC chromatography using fluorescence detection.

3.Progress Report

Using our novel small scale (teaball) malting procedure we malted and analyzed the primary malting quality traits for approximately 1000 samples including 2-row and 6-row types, spring and winter lines, malting and feed quality lines, selections and named varieties. Quality analysis comparisons between the small-scale and standard scale malts showed excellent correlations (r =0.9) for a number of key malting quality attributes such as malt extract, diastatic power, soluble protein and total malt protein. Slightly lower but still very good correlations (0.9 = r = 0.7) were also seen for a-amylase activity, free amino nitrogen, and Kolbach index (soluble/total malt protein). All correlations were significant at p < 0.0001 indicating that the small scale malts should be useful predictors of traditional malting quality metrics, even at the higher sample throughput of the small scale method. This research project addresses objective 2 "Development of novel tools to improve the selection efficiency for identifying superior malting barley varieties" of the current research plan and objective 1 "Identify, develop, validate, and implement new measurements of malting quality, especially those relating to protein mobilization during germination, in barley germplasm in order to identify those genotypes showing enhanced malting quality attributes" in the research plan under NP 306. This research seeks to validate a set of malting and malt quality analysis tools that will add a valuable capability, specifically the ability to malt and analyze limited-availability seed collections for potential malting quality.

Relationship of tocol content to winter hardiness in barley. There is a considerable interest in developing germplasm for winter barley varieties. Recent studies with Arabidopsis have shown a relationship between tocol biosynthesis and enhanced cold tolerance. This project is designed to investigate whether barley lines with enhanced winter hardiness demonstrate a clear quantitative or qualitative difference in their tocol content. This project is being conducted in collaboration with other ARS scientists at Madison Wisconsin and is still in its preliminary stages. Stress conditions resulting in differentiation of cold-tolerant from non-cold tolerant barley are being developed. These experiments will determine if tocol content can be used as a phenotypic marker associated with winter hardiness in barley. Objective: Objective 3: "Evaluate oat and barley germplasm for anti-oxidants and other phytonutrients". Milestones: Provide collaborators analytical data on antioxidant, betaglucan, protein, lipid, etc. in their oat/barley germplasm. The project is monitored through phone calls and email exchanges.